Sequential inactivation of Rho GTPases and Lim kinase by Pseudomonas aeruginosa toxins ExoS and ExoT leads to endothelial monolayer breakdown

Pseudomonas aeruginosa is a major human opportunistic pathogen and one of the most important causal agents of bacteremia. For non-blood-borne infection, bacterial dissemination requires the crossing of the vascular endothelium, the main barrier between blood and the surrounding tissues. Here, we investigated the effects of P. aeruginosa type 3 secretion effectors, namely ExoS, ExoT, and ExoY, on regulators of actin cytoskeleton dynamics in primary endothelial cells. ExoS and ExoT similarly affected the Lim kinase-cofilin pathway, thereby promoting actin filament severing. Cofilin activation was also observed in a mouse model of P. aeruginosa-induced acute pneumonia. Rho, Rac, and Cdc42 GTPases were sequentially inactivated, leading to inhibition of membrane ruffling, filopodia, and stress fiber collapse, and focal adhesion disruption. At the end of the process, ExoS and ExoT produced a dramatic retraction in all primary endothelial cell types tested and thus a rupture of the endothelial monolayer. ExoY alone had no effect in this context. Cell retraction could be counteracted by overexpression of actin cytoskeleton regulators. In addition, our data suggest that moesin is neither a direct exotoxin target nor an important player in this process. We conclude that any action leading to inhibition of actin filament breakdown will improve the barrier function of the endothelium during P. aeruginosa infection.     

Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli

Primary cilia with a diameter of ~200 nm have been implicated in development and disease. Calcium signaling within a primary cilium has never been directly visualized and has therefore remained a speculation. Fluid-shear stress and dopamine receptor type-5 (DR5) agonist are among the few stimuli that require cilia for intracellular calcium signal transduction. However, it is not known if these stimuli initiate calcium signaling within the cilium or if the calcium signal originates in the cytoplasm. Using an integrated single-cell imaging technique, we demonstrate for the first time that calcium signaling triggered by fluid-shear stress initiates in the primary cilium and can be distinguished from the subsequent cytosolic calcium response through the ryanodine receptor. Importantly, this flow-induced calcium signaling depends on the ciliary polycystin-2 calcium channel. While DR5-specific agonist induces calcium signaling mainly in the cilioplasm via ciliary CaV1.2, thrombin specifically induces cytosolic calcium signaling through the IP₃ receptor. Furthermore, a non-specific calcium ionophore triggers both ciliary and cytosolic calcium responses. We suggest that cilia not only act as sensory organelles but also function as calcium signaling compartments. Cilium-dependent signaling can spread to the cytoplasm or be contained within the cilioplasm. Our study thus provides the first model to understand signaling within the cilioplasm of a living cell.     

Glutamate transporters in the biology of malignant gliomas

Malignant gliomas are relentless tumors that offer a dismal clinical prognosis. They develop many biological advantages that allow them to grow and survive in the unique environment of the brain. The glutamate transporters system x _c ^? and excitatory amino acid transporters (EAAT) are emerging as key players in the biology and malignancy of these tumors. Gliomas manipulate glutamate transporter expression and function to alter glutamate homeostasis in the brain, which supports their own growth, invasion, and survival. As a consequence, malignant cells are able to quickly destroy and invade surrounding normal brain. Recent findings are painting a larger picture of these transporters in glioma biology, and as such are providing opportunities for clinical intervention for patients. This review will detail the current understanding of glutamate transporters in the biology of malignant gliomas and highlight some of the unique aspects of these tumors that make them so devastating and difficult to treat.     

Induction of recombination between diverged sequences in a mammalian genome by a double-strand break

To investigate whether mammalian cells can carry out recombinational double-strand break (DSB) repair between highly diverged sequences, mouse fibroblasts were transfected with DNA substrates that contained a “recipient” thymidine kinase (tk) gene disrupted by the recognition site for endonuclease I-SceI. Substrates also contained a linked “donor” tk gene sequence. Following DSB induction by I-SceI, selection for tk-expressing clones allowed recovery of repair events occurring by nonhomologous end-joining or recombination with the donor sequence. Although recombinational repair was most efficient when donor and recipient shared near-perfect homology, we recovered recombination events between recipient and donor sequences displaying 20 % nucleotide mismatch. Recombination between such imperfectly matched (“homeologous”) sequences occurred at a frequency of 1.7 × 10^?7 events per cell and constituted 3 % of the DSB repair events recovered with the pair of homeologous sequences. Additional experiments were done with a substrate containing a donor sequence comprised of a region sharing high homology with the recipient and an adjacent region homeologous to the recipient. Recombinational DSB repair tracts initiating within high homology propagated into homeology in 11 of 112 repair events. These collective results contrasted with our earlier work in which spontaneous recombination (not intentionally induced by a DSB) between homeologous sequences occurred at an undetectable frequency of less than 10^?9 events per cell, and in which events initiating within high homology propagated into adjoining homeology in one of 81 events examined. Our current work suggests that homology requirements for recombination are effectively relaxed in proximity to a DSB in a mammalian genome.     

Critical role of extracellular vesicles in modulating the cellular effects of cytokines

Under physiological and pathological conditions, extracellular vesicles (EVs) are present in the extracellular compartment simultaneously with soluble mediators. We hypothesized that cytokine effects may be modulated by EVs, the recently recognized conveyors of intercellular messages. In order to test this hypothesis, human monocyte cells were incubated with CCRF acute lymphoblastic leukemia cell line-derived EVs with or without the addition of recombinant human TNF, and global gene expression changes were analyzed. EVs alone regulated the expression of numerous genes related to inflammation and signaling. In combination, the effects of EVs and TNF were additive, antagonistic, or independent. The differential effects of EVs and TNF or their simultaneous presence were also validated by Taqman assays and ELISA, and by testing different populations of purified EVs. In the case of the paramount chemokine IL-8, we were able to demonstrate a synergistic upregulation by purified EVs and TNF. Our data suggest that neglecting the modulating role of EVs on the effects of soluble mediators may skew experimental results. On the other hand, considering the combined effects of cytokines and EVs may prove therapeutically useful by targeting both compartments at the same time.     

Mechanisms of cellular invasion by intracellular parasites

Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world’s population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite–host cell interactions, forming the basis of the parasite’s cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.     

A bibliographical study of the introduction of Lavoisier's Traité élémentaire de chimie into Great Britain and America

As the most famous woman scientist of the twentieth century, there has been no shortage of books and articles on the life and career of Marie Curie (1867–1934). Her role as a director of a laboratory-based research school in the new scientific field of radioactivity, a field which embraced both chemistry and physics, however, has never been examined. In recent years, there has been a growing interest in the question of research schools, and Morrell, Ravetz, Geison, and Klosterman, amongst others, have written on this subject. Using, in part, the methodology of Morrell, this paper investigates the role of Marie Curie as a school director in the Paris Faculty in the years 1907–14, examining the work and characteristics of her school and assessing her effectiveness as a director.     

The introduction of the differential notation to Great Britain

In the history of chemistry, the Danish chemist Julius Thomsen (1826–1909) is best known for his contributions to thermochemistry. Throughout his life, he was a pronounced atomist and a tireless advocate of neo-Proutian views as to the constitution of matter. On many occasions, especially in his later years, he engaged in speculations concerning the unity of matter and the complexity of atoms. In this engagement, Thomsen was alone in Danish chemistry, but his works were representative of a large number of 19th-century chemists, particularly in England and Germany. Thomsen's ideas as to the constitution of matter, the periodic system and the noble gases, may be seen as typical of this vigorous trend in fin de siècle chemistry.     

Chemistry and meteorology, 1700–1825

In 1639–1640 Benedetto Castelli (1577–1643) wrote a treatise on the loadstone which is quite unlike any of its contemporaries. In it are the origins of the notion of elementary magnets sharing a common alignment, the idea that all materials are magnetic in different ways, and the first intimation of the conception of magnetic domains. Castelli did not publish his treatise. Nevertheless his work was noted during his life-time, and may have exerted an influence on the development of magnetic theory in the 17th century. The treatise was published in 1883. Since then, however, it has either been neglected or not appreciated. It deserves being rescued from the neglect of more than three centuries.